The present invention relates to derivatives of 2,3,4,4a-tetrahydro-1H-pyrazino[1,2,-a]quinoxalin-5(6H)ones which are serotonin 5-hydroxytryptamine 2C (5HT2C) receptor agonists useful for the treatment of disorders such as obsessive-compulsive disorder, depression, anxiety, schizophrenia, migraine, sleep disorders, eating disorders, obesity, type II diabetes, and epilepsy.
Obesity is a medical disorder characterized by an excess of body fat or adipose tissue. Comorbidities associated with obesity are Type II diabetes, cardiovascular disease, hypertension, hyperlipidemia, stroke, osteoarthritis, sleep apnea, gall bladder disease, gout, some cancers, some infertility, and early mortality. As the percentage of obese individuals continues to rise both in the U.S. and abroad, obesity is expected to be a major health risk in the 21st Century. The serotonin 5-hydroxytryptamine (5-HT) receptor is a G-protein coupled receptor which is expressed in neurons in many regions of the human central nervous system. [Wilkinson, L. 0. and Dourish, C. T. in Serotonin Receptor Subtypes: Basic and Clinical Aspects (ed. Peroutka, S. J.) 147-210 (Wiley-Liss, New York, 1991).] The 5HT2C receptor (formerly called the 5HT1C receptor) is a prominent subtype of the serotonin receptor found in the central nervous system of both rats and humans. It is expressed widely in both cortical and subcortical regions. [Julius, D. MacDermott, A. B., Axel, R. Jessell, T. M. Science 241:558-564 (1988).] Studies in several animal species and in humans have shown that the non-selective 5HT2C receptor agonist, meta-chlorophenylpiperazine (MCPP) decreases food intake. [Cowen, P. J., Clifford, E. M., Williams, C., Walsh, A. E. S., Fairburn, C. G. Nature 376: 557 (1995).] Tecott, et al have demonstrated that transgenic mice lacking the 5HT2C receptor eat more and are heavier than Wild Type mice. [Tecott, L. H., Sun, L. M., Akana, S. F., Strack, A. M., Lowenstein, D. H., Daliman, M. F., Jullus, D. Nature 374: 542-546 (1995).] Compounds of this invention are 5HT2C receptor subtype selective agonists which are selective over other monoamine receptors, causes a reduction in food intake and result in a reduction in weight gain. Other therapeutic indications for 5HT2C agonists are obsessive compulsive disorder, depression, panic disorder, schizophrenia, sleep disorders, eating disorders, and epilepsy.
U.S. Pat. Nos. 4,032,639; 4,089,958; and 4,203,987 describe 2,3,4,4a-Tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6)-ones and derivatives thereof as antihypertensive agents. In contrast, compounds of this invention bind to and activate the 5HT2C receptors in the CNS and are useful for the treatment of CNS disorders.
Indian J. Chem. 17B, 244-245 (1979) discloses 3-Substituted 2,3,4,4a,5,6-Hexahydro-1 (H)-pyrazino[1,2-a]quinoxalines which exhibit no anorexigenic or stimulant activity at 60 mg/kg i.p. dose. Weak CNS depressant activity and significant hypotensive activity in anaesthetized animals. Tachyphylaxis was observed.
This invention provides compounds of formula I having the structure 
wherein
R is hydrogen or alkyl of 1-6 carbon atoms;
Rxe2x80x2 is hydrogen, alkyl of 1-6 carbon atoms, acyl of 2-7 carbon atoms, or aroyl;
R1, R2, R3, and R4 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, halogen, trifluoroalkyl, xe2x80x94CN, alkyl sulfonamide of 1-6 carbon atoms, alkyl amide of 1-6 carbon atoms, amino, alkylamino of 1-6 carbon atoms, dialkylamino of 1-6 carbon atoms per alkyl moiety, trifluoroalkoxy of 1-6 carbon atoms, acyl of 2-7 carbon atoms, or aroyl;
X is CR5R6 or a carbonyl group;
R5 and R6 are each, independently, hydrogen or alkyl of 1-6 carbon atoms;
or a pharmaceutically acceptable salt thereof, with the proviso that at least one of R1, R2, R3, or R4 are not hydrogen;
which are 5HT2C receptor agonists useful for the treatment of disorders involving the central nervous system such as obsessive-compulsive disorder, depression, anxiety, panic disorder, schizophrenia and schizophrenic disorders, migraine, sleep disorders, eating disorders, obesity, type II diabetes, and epilepsy.
This invention provides methods for treatment, inhibition or alleviation of symptoms of schizophrenic disorders in a mammal in need of such treatment. These methods include those for the schizophrenic disorders known in the art, including those defined by the American Psychiatric Associations Diagnostic and Statistical Manual of Mental Disorders, Third Edition (DSM-III, 1980), its revision DSM-III-R (1987) or the DSM-IV (1996). These methods include those for schizophrenia, schizophreniform syndromes, or schizoaffective disorders. Also included are related psychotic syndromes referred to as brief reactive psychoses, as well as borderline or latent schizophrenia and simple schizophrenia, which are also referred to as borderline or schizotypal personality disorders. Additional methods include late onset schizophrenia-like syndromes, such as the involutional paraphrenias, which are also known as paranoid disorder or atypical psychosis.
The compounds of this invention may contain an asymmetric carbon atom and some of the compounds of this invention may contain one or more asymmetric centers and may thus give rise to optical isomers and diastereoisomers. While shown without respect to stereochemistry in Formula I, the present invention includes such optical isomers and diastereoisomers; as well as the racemic and resolved, enantiomerically pure R and S stereoisomers; as well as other mixtures of the R and S stereoisomers and pharmaceutically acceptable salts thereof.
The term xe2x80x9calkylxe2x80x9d includes both straight- and branched-chain saturated aliphatic hydrocarbon groups. The term xe2x80x9caroylxe2x80x9d is defined as an aryl ether, where aryl is defined as an aromatic system of 6-14 carbon atoms, which may be a single ring or multiple aromatic rings fused or linked together as such that at least one part of the fused or linked rings forms the conjugated aromatic system. Preferred aryl groups include phenyl, naphthyl, biphenyl, anthryl, tetrahydronaphthyl, phenanthryl groups. Halogen is defined as Cl, Br, F, and I.
Pharmaceutically acceptable salts can be formed from organic and inorganic acids, for example, acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic, camphorsulfonic, and similarly known acceptable acids.
Preferred compounds of this invention are those in which at least one of R1, R2, R3, or R4 are not hydrogen, and the non-hydrogen substituents of R1, R2, R3, and R4 are halogen or trifluoromethyl.
Preferred enantiomerically pure compounds of formulas IA and IB are provided as follows: 
wherein R, R1, R2, R3, and R4 are as described above.
The compounds of this invention can be prepared according to the following schemes from commercially available starting materials or starting materials which can be prepared using literature procedures. These schemes show the preparation of representative compounds of this invention. 
In Scheme 1, the symbol Cbz represents a carbobenzyloxy group and Y stands for chlorine, fluorine, or bromine. A solution of 4-carbobenzyloxypiperazine-2-carboxylic acid (I) is allowed to react with a substituted ortho-nitrohalobenzene (II) to give a 4-carbobenzyloxy-1-(o-nitro-substituted-phenyl)-piperazine-2-carboxylic acid (III). The reaction is carried out in an inert organic solvent, such as dimethylsulfoxide, in the presence of a base, such as triethylamine, at a temperature above ambient temperature, such as 50-150xc2x0 C.
The intermediate (III) is cyclized by a process involving reduction of the nitro group to an amino group, preferably by reaction of a metal, such as iron, in an acid, such as acetic acid, followed by heating at elevated temperature, such as 50-100xc2x0 C., to effect cyclization to (IV). Removal of the Cbz protecting group using boron tribromide, catalytic reduction or a base, such as potassium hydroxide, gives products of this invention (IVa). Or treatment of (IV) with a base, such as sodium hydride, followed by reaction with an alkyl halide, such as methyl iodide, give intermediates (V). Removal of the Cbz group with boron tribromide or potassium hydroxide give compounds of this invention (VI) where Rxe2x80x2 is lower alkyl.
Compounds (VI) can also be alkylated a second time using a base, such as sodium hydride, and an alkyl halide, such as methyl iodide, to give compounds of this invention (VIII). Alternatively, compounds (VI) can be reduced with a reducing agent, such as borane in THF, to compounds of this invention (VII). Compounds (VII) can also be reduced with borane in THF to give (IX) which are compounds of this invention.
The amide of compounds (V) can also be reduced to amines (VII) using a reducing agent, such as borane in tetrahydrofuran, at 0-50xc2x0 C. Compounds (VII) are also compounds of this invention.
Likewise, the amide of compounds (VI) can be reduced to amines (VII) which are compounds of this invention. In compounds (VI) where Rxe2x80x2 is acyl this group is put on, as already described, after reduction of amides (VI) where Rxe2x80x2 is hydrogen. 
In Scheme 2, the carboxylic acids of intermediate (IV) are converted to the corresponding N-methoxy-N-methyl amides (IX) by reaction of the corresponding acids (IV) with N,O-dimethylhydroxylamine hydrochloride in the presence of a base, such as pyridine, and a coupling reagent, such as dicyclohexylcarbodiimide (DCC) in an organic solvent, such as methylene chloride at a temperature between 0-50xc2x0 C. Treatment of intermediates (IX) with Grignard reagents or organolithium reagents, such as methyl lithium, gives ketones (X). Reduction of the nitro group in intermediates (X) with a reducing agent, such as iron in acetic acid, gives the corresponding amines (XI) which cyclize at elevated temperatures, such as 50-150xc2x0 C., in the presence of an acid, such as p-toluenesulfonic acid, in an inert organic solvent, such as benzene, to give compounds of this invention (XII). Alkylation of (XII) with an alkyl halide, such as methyl iodide, or an acyl halide such as acetyl chloride, gives compounds of this invention (XIII). Treatment of intermediates (X) with a Grignard reagent, such as methylmagnesium chloride, give tertiary alcohols (XIV). Reduction of the nitro group in intermediates (XIV) with a metal, such as iron, in an acid, such as acetic acid, followed by heating at a temperature from 50-150xc2x0 C., gives (XV) which are compounds of this invention. Reaction of compounds (XV) with an alkyl halide, such as methyl iodide, or an acyl halide, such as acetyl chloride, gives (XVI) which are compounds of this invention.
The enantiomerically pure compounds of this invention can be prepared according to the following Scheme 3 from commercially available starting materials or starting materials which can be prepared using literature procedures. This scheme shows the preparation of representative (R)-compounds of formula IA of this invention, starting with the known 2-(R)-piperazinecarboxylic acid (reference below). Starting from the known 2-(S)-piperazinecarboxylic acid gives the (S)-compounds of formula IB of this invention. 
In Scheme 3, the (R)-2-piperazinecarboxylic acid (prepared according to the references below) was converted by standard methods to the N-protected amino-acid (XVII); the symbol Cbz represents a carbobenzyloxy group. A solution of (R)-4-carbobenzyloxypiperazine-2-carboxylic acid (XVII) is allowed to react with a substituted ortho-nitrofluorobenzene to give a 4-carbobenzyloxy-1-(o-nitro-substituted-phenyl)-(R)-piperazine-2-carboxylic acid (XVIII). The reaction is carried out in an inert organic solvent, such as dimethylformamide, in the presence of a base, such as triethylamine, at a temperature above ambient temperature, such as 50-70xc2x0 C.
The intermediate (XVIII) is cyclized by a process involving reduction of the nitro group to an amino group, preferably by reaction of a metal, such as iron, in an acid, such as acetic acid, followed by heating at elevated temperature, such as 50-70xc2x0 C., to effect cyclization to (XIX). Removal of the Cbz protecting group using 30% HBr in acetic acid, boron tribromide, or catalytic reduction, gives chiral products of this invention (IA).
The ability of the compounds of this invention to act as 5HT2C agonists was established is several standard pharmacological test procedures; the procedures used and results obtained are provided below.
Test Procedures
5HT2C Receptor Binding Test Procedure
To evaluate high affinity for the 5HT2C receptor, a CHO (Chinese Hamster Ovary) cell line transfected with the cDNA expressing the human 5-hydroxy-tryptamine2C (h5HT2C) receptor was maintained in DMEM (Dulbecco""s Modified Eagle Media) supplied with fetal calf serum, glutamine, and the markers: guaninephosphoribosyl transferase (GTP) and hypoxanthinethymidine (HT). The cells were allowed to grow to confluence in large culture dishes with intermediate changes of media and splitting. Upon reaching confluence, the cells were harvested by scraping. The harvested cells were suspended in half volume of fresh physiological phosphate buffered saline (PBS) solution and centrifuged at low speed (900xc3x97g). This operation was repeated once more. The collected cells were then homogenized with a polytron at setting #7 for 15 sec in ten volumes of 50 mM Tris.HCl, pH 7.4 and 0.5 mM EDTA. The homogenate was centrifuged at 900xc3x97g for 15 min to remove nuclear particles and other cell debris. The pellet was discarded and the supernatant fluid recentrifuged at 40,000xc3x97g for 30 min. The resulting pellet was resuspended in a small volume of Tris.HCl buffer and the tissue protein content was determined in aliquots of 10-25 microliter (xcexcl) volumes. Bovine Serum Albumin (BSA) was used as the standard in the protein determination by the method of Lowry et al., (J. Biol. Chem., 193:265 (1951). The volume of the suspended cell membranes was adjusted with 50 mM Tris.HCl buffer containing: 0.1% ascorbic acid, 10 mM pargyline and 4 mM CaCl2 to give a tissue protein concentration of 1-2 mg per ml of suspension. The preparation membrane suspension (many times concentrated) was aliquoted in 1 ml volumes and stored at xe2x88x9270xc2x0 C. until used in subsequent binding experiments.
Binding measurements were performed in a 96 well microtiter plate format, in a total volume of 200 xcexcl. To each well was added: 60 xcexcl of incubation buffer made in 50 mM Tris.HCl buffer, pH 7.4 and containing 4 mM CaCl2; 20 xcexcl of [125I] DOI (S.A., 2200 Ci/mmol, NEN Life Science).
The dissociation constant, KD of [125I] DOI at the human serotonin 5HT2C receptor was 0.4 nM by saturation binding with increasing concentrations of [125I] DOI. The reaction was initiated by the final addition of 100.0 xcexcl of tissue suspension containing 50 xcexcg of receptor protein. Nonspecific binding is measured in the presence of 1 xcexcM unlabeled DOI added in 20.0 xcexcl volume. Test compounds were added in 20.0 ml. The mixture was incubated at room temperature for 60 min. The incubation was stopped by rapid filtration. The bound ligand-receptor complex was filtered off on a 96 well unifilter with a Packard(copyright) Filtermate 196 Harvester. The bound complex caught on the filter disk was dried in a vacuum oven heated to 60xc2x0 C. and the radioactivity measured by liquid scintillation with 40 xcexcl Microscint-20 scintillant in a Packard TopCount(copyright) equipped with six (6) photomultiplier detectors.
Specific binding is defined as the total radioactivity bound less the amount bound in the presence of 1 xcexcM unlabeled DOI. Binding in the presence of varying concentrations of test drugs is expressed as percent of specific binding in the absence of drug. These results are then plotted as log % bound vs log concentration of test drug. Non linear regression analysis of data points yields both the IC50 and the Ki values of test compounds with 95% confidence limits. Alternatively, a linear regression line of decline of data points is plotted, from which the IC50 value can be read off the curve and the Ki value determined by solving the following equation:   Ki  =            IC      ⁢              xe2x80x83            ⁢      50              1      +              L        /        KD            
where L is the concentration of the radioactive ligand used and the KD is the dissociation constant of the ligand for the receptor, both expressed in nM.
The following Ki""s are provided for various reference compounds:
Stimulation of [3H] Inositol Monophosphate Production by 5HT2C Agonists.
CHO cells transfected with the cDNA expressing the human 5-HT2C receptor were cultured in Dulbecco""s modified Eagle""s medium (DMEM) supplemented with 10% fetal bovine serum and non-essential amino acids. Upon reaching confluence the cells were harvested using PBS/EDTA and plated in 24 well plates at an initial density of 2.5xc3x97105 cells per well. One (1) ml of maintenance medium containing 1 xcexcCi/ml myo-[3H] inositol was added to each well. After 48 hours labeling, the cells were washed once with 0.5 ml DMEM containing 25 mM HEPES and 10 mM LiCl, then preincubated with the medium for 30 min (antagonists were included in this period if tested). At the end of the preincubation, the medium was removed, the cells were then incubated with test compounds (in presence of antagonists if needed) for 30 min. The reaction was terminated by removal of the incubation solution and addition of 0.5 ml ice-cold 5% PCA, followed by 15 to 30 min incubation on ice. 200 xcexcl of 0.5 M Tes/1.5 M K2CO3 was added to each well to neutralize to pH 7, and plates were left on ice for another 15 to 30 min to precipitate all salts. The liquid and solid phases were separated by centrifugation.
A portion (350 xcexcl) of the upper aqueous phase was applied to Dowex AG-1X8 (formate form, 100-200 mesh) columns. The columns were then washed stepwise with 10 ml of water and 10 ml of 25 mM ammonium formate to remove free myo-[3H]inositol and deacylated phosphoinositol, respectively. Finally 10 ml of 0.2 M ammonium formate solution was applied to the columns to elute [3H] inositol monophosphate ([3H] IP1) directly into scintillation vials. Of this eluate, 1 ml was used to determine radioactivity by scintillation counting.
Agonist-stimulated levels of [3H]inositol monophosphate (IP1) is expressed as a percentage of the response observed with a maximally effective concentration of 5-HT (10 xcexcM). A 3-parameter logistic function is used to generate estimate of EC50/IC50. Antagonists are tested in the presence of 10 xcexcM 5-HT.
The following data are provided for various reference compounds:
Effects of Compounds on Feeding Behavior in Rats
Eight (8) male Sprague-Dawley rats weighing 150-180 g were separated into individual cages and acclimated to a powdered diet for 2 weeks. During this period and throughout the test procedure, the food cup and the animals were weighed daily. Following the acclimation period, animals were fasted for 24 hours and then injected with either vehicle or one of 4 doses of the test compound. Food intake was assessed at 2 and 24 hours following compound administration. Compounds to be evaluated were injected 1-2xc3x97 per week until all animals had received all doses of the test compound. The order of doses were chosen using to a modified Latin Square design. Additional studies may be conducted in satiated rats at the start of the dark cycle. Compounds were injected i.p, s.c. or p.o. At the end of the study effects of the test compound on food intake was evaluated using a repeated measures ANOVA. Data were collected were 2 hour food intake (g). Data were subjected to one-way ANOVA with posthoc t-tests to assess group differences. Where appropriate, ED50 values were calculated. The ED50 value is the dose that produces a 50% reduction in food intake during the test period.
Results
The results obtained in this standard pharmacological test procedures demonstrate that the compounds of this invention are 5HT2C receptor agonists useful for the treatment of diseases involving the central nervous system such as obsessive-compulsive disorder; depression; anxiety; panic disorder; schizophrenia; migraine; sleep disorders, such as sleep apnea; eating disorders, such as hyperphagia; obesity; type II diabetes; and epilepsy.
The compounds of this invention can be formulated neat or with a pharmaceutical carrier for administration, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmacological practice. The pharmaceutical carrier may be solid or liquid.
A solid carrier can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, lecithins, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.
Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. The compounds of this invention can also be administered orally either in liquid or solid composition form.
The compounds of this invention may be administered rectally or vaginally in the form of a conventional suppository. For administration by intranasal or intrabronchial inhalation or insufflation, the compounds of this invention may be formulated into an aqueous or partially aqueous solution, which can then be utilized in the form of an aerosol. The compounds of this invention may also be administered transdermally through the use of a transdermal patch containing the active compound and a carrier that is inert to the active compound, is non toxic to the skin, and allows delivery of the agent for systemic absorption into the blood stream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may also be suitable. A variety of occlusive devices may be used to release the active ingredient into the blood stream such as a semipermeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient. Other occlusive devices are known in the literature.
The dosage requirements vary with the particular compositions employed, the route of administration, the severity of the symptoms presented and the particular subject being treated. Based on the results obtained in the standard pharmacological test procedures, projected daily dosages of active compound would be 0.02 xcexcg/kg-750 xcexcg/kg. Treatment will generally be initiated with small dosages less than the optimum dose of the compound. Thereafter the dosage is increased until the optimum effect under the circumstances is reached; precise dosages for oral, parenteral, nasal, or intrabronchial administration will be determined by the administering physician based on experience with the individual subject treated. Preferably, the pharmaceutical composition is in unit dosage form, e.g. as tablets or capsules. In such form, the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient; the unit dosage forms can be packaged compositions, for example, packaged powders, vials, ampoules, pre filled syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form.
The following provides the preparation of a representative compound of this invention.